Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
The interferons (IFNs) comprise a group of glycoproteins produced by various cells in response to viral infections, specific antigens or mitogens. Since their discovery (1), the IFNs have been found to play important roles in, for example, antiviral, anti-proliferative, differentiative and immunomodulatory responses (2).
Following binding of IFNs to their receptor, intracellular tyrosine kinases of the JAK family become activated and phosphorylate transcription factor molecules called Stats (Signal transducers and activators of transcription). IFN-gamma signals by activating the formation of a homodimer of Stat 1. IFN-alpha or beta mainly signal by activating the interferon sensitive gene factor 3 (ISGF3), a complex of Stat1 and/or Stat2, as homo or heterodimers that combine to produce trimeric molecules containing a third protein, p48 ISGF3-gamma. After activation, the transcription factor complexes migrate to the nucleus, binding to target DNA sequences thereby affecting the expression of interferon sensitive genes (ISGs). The Stat1 homodimer binds a sequence TTCNNNGAA, known as the GAS site. IFN signalling also activates one other interferon regulator factor, IRF-1. Both factors, IRF-1 and ISGF3 bind via regulatory response elements in the promoter regions of ISGs that comprise direct repeats of the DNA sequence GAAANN, leading to activation of transcription from the IGS (for reviews, see (3, 4)).
The genes encoding IFNs and components of the IFN signalling pathway are proposed to belong to a family of tumour suppressor genes (5). Accordingly, mutations disrupting any steps in the IFN signal transduction pathway would be expected to reduce cellular responsiveness to IFN, abrogating the tumour suppressive function of IFN action and thereby facilitating the onset of cancer.
The effect of IFN in the treatment of advanced malignant melanoma has been demonstrated in several clinical trials. In this regard, IFN-alpha is effective in only a group (˜23%) of skin melanoma patients. An explanation was proposed for this phenomenon based on abnormalities detected in the IFN signalling properties of melanoma cells (6-8). Studies on growth of short-term cultures at low passage number established from advanced stage III metastatic melanomas revealed all patients' samples to be resistant to IFN when compared to IFN-sensitive melanoma cell lines and melanocytes (6). As a result, the majority of patients with advanced metastatic melanomas will fail to respond significantly to IFNs because their tumour cells fail to adequately respond to the direct actions of the IFNs. IFNs act in vivo in two ways to either indirectly stimulate immune effector cells or directly act on the tumour cell targets (9). Studies of tumour cell lines produced in knockout mice which have been made IFN-insensitive (by using either Stat1-deficient or IFN-gamma receptor-deficient mice) have revealed that the direct effects of IFN are important for immune surveillance (10). Cancer cells established in the IFN-gamma insensitive mice, when passaged into syngeneic wild type mice, were no longer rejected by the immune system. Thus, it was concluded that IFN action was mediated in part through its direct effects on the tumour cells, presumably by inducing enhanced tumour cell immunogenicity (10). Thus, loss of responsiveness to IFNs as tumour suppressors is one of the early and important developments in the onset of malignancy as it allows tumours to evade immune surveillance, providing the tumours with a significant survival advantage.
Many studies have reported various defects in the IFN system as being responsible for the different sensitivities to type I IFNs in cell lines established from other tumour types: (i) IFN-alpha/-beta gene deletion in acute leukaemia cell lines (11) and malignant T cells (12); (ii) alteration or down regulation of IFN-alpha receptor gene expression in hairy cell leukaemia (13) and lymphoblastoid cells (14); (iii) interference with the induction of the expression of IFN-stimulated genes in B lymphoid cell lines (15) and Burkitt's lymphoma cells (16); (iv) defects in the activation of transcription factors in Daudi cells (17) and primary leukaemia cells (18). In addition, melanoma cell lines with a wide variation in their responsiveness to the anti-proliferative (19) and antiviral (20) activities of IFNs, ranging from highly sensitive to relatively resistant, have been described.
Initial studies by the present inventors on IFN signalling in melanoma cell lines with different responsiveness to IFN revealed that these cell lines did not show significant differences in the levels of IFN binding to cell surface receptors (21) or in the activation of the IFN receptor associated JAK tyrosine kinases (7). Thus, loss of IFN responsiveness did not appear to be due to abnormalities in the IFN mediated activation of receptor signalling. However, in all IFN-resistant melanoma cells examined, deficiencies were detected at the next level in the IFN activated signalling pathways (6). In this regard, much lower intracellular levels of the trans-activating relay factors essential for transmitting the IFN signal from the membrane to the cell nucleus were detected [for review, see 8]. Hence, changes in gene expression normally induced by IFNs and which are essential if tumours are to be recognized and eliminated by the body's immune system will not occur in the IFN-resistant tumour cells because the signal reaching the nucleus is insufficient.
Cellular responsiveness to IFNs can be increased by prior treatment with IFN (22). This process, called “priming” (6), increases the levels of the cognate transcription factors, including IRF-1 and all three components of ISGF3: Stat1; Stat2; and p48. Regulation of the IRF-1, Stat2 and p48 promoters have been described (for example, see (23) and expression of these genes is regulated by the IFNs. Stat1 protein is involved in activating expression of IRF-1, p48 and Stat2.
Stat1 is at a pivotal point in IFN signalling as it is required for both type I and II IFN receptor signals (3). The regulation of Stat1 activity has become an important biological question for other reasons as well, given other key roles for Stat1 in important cell functions. Thus, IFNs act via Stat1 to regulate cell growth by directly inducing expression of the cell cycle inhibitor, CKI p21 WAF (24, 25). In addition, Stat1 regulates expression of caspases 1, 2 and 3 involved in apoptosis (26).
In work leading up to the present invention, the inventors' analyses of human melanoma cells revealed that Stat1 (6, 7) and IRF-1 were consistently deficient in IFN-resistant melanoma cells. In particular, the evidence pointed to a key role for the transcription factor, Stat1, which was poorly expressed at the mRNA and protein levels in IFN-resistant cells and moreover, responsiveness to IFN was greatly increased by transfecting IFN-resistant melanoma cells to express increased levels of Stat1 (6). Similar results have been shown to occur in breast cancer cell samples as well (27, 28) raising the possibility of a more widespread problem, which highlights the broader significance of Stat1 deficiency to cancers in general. Consistent with this hypothesis, several studies have since reported other cancer types in which Stat1 deficiencies have been commonly noted (29, 30).
The present inventors also found that by treating melanoma cells with high levels of IFN-gamma (gamma-priming), the treated cells express markedly increased levels of Stat1 as well as of p48 and Stat2 (6). Even in type I IFN-resistant melanoma cells, Stat1 levels were increased after gamma-priming. In addition, the cellular responsiveness to treatment with type I IFN was increased after gamma-priming, including significantly greater ISGF3 binding activity detected by electrophoretic gel mobility shift assay (EMSA) and increased induction of ISGs detected by immunochemical methods. Amongst the ISGs whose expression was increased were the IFN inducible surface antigens, class I MHC and ICAM-1 (6), which are important in immune cell recognition of tumour cells.